A. Field Of The Invention
The present invention is directed to a trivalent coupling agent. More particularly, the present invention is directed to a tris-maleimido compound having three linker arms of variable length, charge, lability and hydrophobicity. Such compounds are useful as intermediates in the synthesis of bifunctional and trifunctional (i.e., tri-specific) antibody-like compounds which are useful in medical diagnoses, therapeutics and diagnostic/therapeutic combinations.
B. Background
Antibodies are complex protein molecules generated by an organism's immune system in response to an antigen perceived by the host as being foreign. The extreme plasticity and diversity of an animal's immune repertoire permits the generation of an enormous variety of antibody molecules to an equally large number of antigens. However, individual antibodies are monospecific and therefore merely monofunctional for purposes of this invention.
Landsdorp, et al. teaches the formation of a bifunctional antibody complex formed by cross linking two monoclonal antibodies of different specificities but of the same isotype. Landsdorp, et al., "Cyclic Tetramolecular Complexes Of Monoclonal Antibodies: A New Type Of Cross-linking Agent," Eur. J. Immunol., 16, p. 679-83 (1986). Landsdorp's cross-linking agent consisted of two anti-isotype antibody molecules, which cross-linked the two monoclonal antibodies to form a cyclic tetramolecular complex that was bifunctional. Implicitly, the intact antibodies taught by Landsdorp have Fc regions which are capable of binding complement and/or stimulating an immune response if presented in vivo.
An object of the present invention is to avoid the use of antibodies or linkers having Fc regions which may bind, complement, and/or stimulate an immune response by the antibody-target complex.
Reading (U.S. Pat. No. 4,714,681) teaches the creation of bifunctional antibody-antibody chimeras by the fusion of two different hybridoma cell lines which produce monoclonal antibodies of different specificities (quadroma) and by the fusion of a hybridoma producing a specific monoclonal antibody with a lymphocyte producing a different antibody. The success of this method depends on the ability of the hybrid cells to produce both the heavy and light chains of both parental types in equal amounts such as to maximize the potential for the random assembly of heavy and light chains to yield the appropriate bifunctional complex. At best, this random assembly of antibody subunits can result in only one of eight molecules (12.5%) being of the desired specificities. Moreover, Reading's chimeras are whole antibodies that have intact Fc regions. Consequently, when injected into a "foreign" species, Reading's chimeras, like Langsdorp's bifunctionals, have the potential to invoke an interaction with components of the immune system that bear Fc receptors (e.g., macrophages, complement, etc.).
Among the first descriptions of the use of chemical compounds to covalently cross-link antibodies was Hamaguchi et al., J. Biochem, 85; 1289-1300 (1979). Hamaguchi describes the synthesis of a bifunctional-antibody-.beta.-galactosidase compound which is cross-linked via N,N'-o-phenylenedimaleimide. Hamaguchi's compound was reported useful in sandwich enzyme immunoassays. Glennie et al., J. Immunol., 139, 2367-2375 (1987), also describes the linking of two Fab' fragments utilizing the compound taught in Hamaguchi, i.e., o-phenylenedimaleimide.
Notwithstanding their characterization as bifunctional, when used as pharmaceutical agents, the bifunctional antibodies and bifunctional Fab' compounds of the prior art have the inherent limitation of being monofunctional at their site of action. This limitation arises because the first of the two specificities of the bifunctional molecule must be directed to the site of action, i.e., the organ, tissue or antigen of interest. This leaves only a single specificity for conferring mono-function to the molecule once it has become immobilized at its site of action. It is an object of the present invention to develop an intermediate compound, i.e., a multifunctional coupling agent, that is suited for producing a novel series of pharmaceutical agents capable of being trifunctional overall, and thus bifunctional at their site of action.